1. Field of the Invention
This invention relates to a method for purifying human tissue plasminogen activator (hereinafter referred to as tPA). More specifically, this invention relates to a method for isolating and purifying tPA having a molecular weight of about 70,000 daltons by bringing a crude tPA preparation containing tPA and other undesired proteins into contact with a cation-exchange resin.
2. Description of the Prior Art
tPA is a protein which has a molecular weight of about 70,000 daltons and is produced in a tissue of a higher animal, serves to activate plasminogen, a precursor of plasmin which is a proteolytic enzyme specific to fibrin.
In order to produce tPA for the pharmaceutical use, it is necessary to sufficiently remove undesired heteroantigenic proteins. If tPA is contaminated with such undesired proteins, these proteins exert the antigenicity in the human body to causes side effects such as anaphylactic shock when administered.
Types of undesired heteroantigenic proteins incorporated as contaminants in the manufacturing processes vary depending on method of cultivation of cells and purification procedure used. In the manufacturing procedure which includes a process of cultivating cells capable of producing tPA, a large amount of undesired heteroantigenic proteins are contained in resultant culture fluids, as constituents of a medium solution and as excretions by the cells.
Representative methods for cultivation include that with the use of a medium containing fetal calf serum and that with the use of a serum-free medium.
In the case where fetal calf serum is used for cultivation, the fetal calf serum contains a group of undesired heteroantigenic proteins which exert antigenicity to human. Proteins derived from fetal calf serum vary but most of them have the pI (isoelectric point) in the range between 4 and 6.
Furthermore, in the case where a serum-free medium is used, substances required for cultivation must be added to the medium depending on the cells used.
Among the substances required for cultivation, as to proteins, hormones, such as insulin, and transferrin are frequently used. The pI values of insulin and transferrin are approximately in the range between 5 and 6.
tPA is known to have two molecular forms, single-chain tPA and double-chain tPA. It is known that, particularly in a method for preparing single-chain tPA, protease inhibitors such as aprotinin are added to a medium during cultivation of cells for the production of tPA.
The protease inhibitors used are also undesired heteroantigenic proteins. The pI of aprotinin is in the range 10-10.5.
Furthermore, tPA containing fluids obtained by partially purifying the above culture fluids by the manner described below can also include undesired proteins.
For the purification of tPA, various types of affinity chromatography have been used. Known examples include concanavalin A-Sepharose (Rijken, D. C. and Collen, D. (1981) J. Biol. Chem. 256, 7035-7041), erythrina trypsin inhibitor (ETI)-Sepharose (Heussen, C., et al. (1984) J. Biol. Chem. 259, 11635-11638), anti-tPA and antibody-Sepharose (Ranby M., et al. (1982) FEBS Lett 146, 289-292) and fibrin-Sepharose (U.S. Pat. No. 4,505,893). Outflow of these immobilized proteins, though in a small amount, is observed during operation. Also, these proteins are undesired heteroantigenic proteins. The ranges of the pI of these proteins are 4.4 to 5.5 for concanavalin, 4.5 to 5.5 for ETI, 5.8 to 7.3 for immunoglobulin G and 5.5 to 5.8 for fibrinogen.
In view of methods for preparing tPA, the present inventors had been intensively investigated means for removing these potential heteroantigenic proteins. As a result, the inventors found that a method with the use of cation-exchange resins is particularly advantageous, and thus completed the present invention.
The use of a cation-exchanger for purification of tPA is known (Japanese Patent Laid-Open No. 174727/1985), in which, however, the cation-exchanger is used for recovering tPA fractions for the purpose of partial purification, which is different from the use of the cation-exchanger in the present invention.